of viral DNA but also resulted in the production of viral particles that exhibited reduced reverse transcriptase activity ... HIV-1 integrase protein were constructed and analyzed for .... viral p24 antigen was produced by cells infected with the S81I.
JOURNAL OF VIROLOGY, Dec. 1994, p. 8401-8405
Vol. 68, No. 12
0022-538X/94/$04.00+0 Copyright © 1994, American Society for Microbiology
Integrase Mutants of Human Immunodeficiency Virus Type 1 with a Specific Defect in Integration BRUNELLA TADDEO,1t WILLIAM A. HASELTINE,2 AND CHRIS M. FARNET'* Division of Human Retrovirology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115,1 and Human Genome Sciences, Inc., Rockville, Maryland 208502 Received 7 June 1994/Accepted 23 August 1994
A previous genetic analysis of the human immunodeficiency virus type 1 integrase protein failed to identify single amino acid substitutions that only block the integration of viral DNA (C.-G. Shin, B. Taddeo, W. A. Haseltine, and C. M. Farnet, J. Virol. 68:1633-1642, 1994). Additional substitutions of amino acids that are highly conserved among retroviral integrases were constructed in human immunodeficiency virus type 1 and analyzed for their effects on viral protein synthesis and processing, virion morphology, and viral DNA synthesis and integration in an attempt to identify mutants with a specific defect in integration. Four single amino acid substitutions resulted in replication defective viruses. Conservative, single amino acid substitutions of the two invariant aspartic acid residues found in all retroviral integrases prevented the integration of viral DNA and had no detectable effect on the other stages in the viral replication cycle, indicating that these mutants exhibited a specific defect in integration. Mutations at two positions, S-81 and P-109, blocked the integration of viral DNA but also resulted in the production of viral particles that exhibited reduced reverse transcriptase activity, suggesting additional defects in viral replication. Substitution of the highly conserved amino acid T66 had no effect on viral replication in a CD4+ human T-cell line. This analysis extends the range of possible phenotypes that may be produced by single amino acid substitutions in conserved residues of the integrase protein.
The integration of a DNA copy of the retroviral RNA into the host cell genome is essential for viral replication. The viral integrase protein, encoded by the 3' end of the viral pol gene, is required for integration (1, 3, 4, 17, 18, 20). Comparison of the amino acid sequences of a number of retroviral integrases has demonstrated the presence of highly conserved residues and motifs that may be important for integrase function (7, 9). The most highly conserved region of retroviral integrases lies in the central region of the protein and is defined by three acidic amino acid residues in a
integrase mutants were constructed by introducing single amino acid substitutions in the highly conserved D,D(35)E region of the protein. The nature and location of each point mutation are indicated in the legend to Fig. 1. The integrase mutants were analyzed for their effects on viral replication by monitoring multiple steps in the viral replication cycle. Each of the integrase mutations was introduced into an HXBc2 provirus, and wild-type and mutant proviral DNAs were transfected into COS-1 cells. Forty-eight hours following transfection, the cells were metabolically labeled with [35S]cysteine, and viral proteins were immunoprecipitated from cell lysates by using an HIV-1-infected patient serum. As shown in Fig. 1, the pattern of viral proteins produced by the integrase mutants was identical to that produced by wild-type virus, demonstrating that none of the amino acid substitutions had detectable effects on the synthesis or processing of viral proteins. Virion release from COS-1 cells transfected with wild-type and integrase mutant proviruses was measured 48 h after transfection by assaying culture supernatants for particleassociated viral proteins, viral p24 antigen, and reverse transcriptase (RT) activity. A wild-type pattern of [35S]cysteinelabeled viral proteins was found in the supernatants of cells transfected with all of the integrase mutants (Fig. 1), indicating that the mutations had no detectable effect on the process of virion formation. Consistent with this observation was the demonstration that each of the integrase mutants released wild-type levels of particle-associated p24 antigen into the culture supernatants, as measured by a radioimmunoassay (data not shown). Surprisingly, supernatants recovered from COS-1 cells transfected with mutants S811 and P109S consistently showed a reduced level of particle-associated RT activity (Fig. 2), even though essentially wild-type levels of viral particles were detected in culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
conserved spatial arrangement [the D,D(35)E motifl (9, 10). Mutations in any one of the three residues result in the
complete loss of all integrase enzymatic activities in vitro (5, 6, 10, 12, 13, 24) and can block viral replication in cell cultures (12, 22). Establishing a correlation between specific in vitro enzymatic defects and the resulting virus replication phenotype is complicated by the pleiotropic effects of some integrase point mutants on viral replication. A previous analysis of single amino acid substitutions in the human immunodeficiency virus type 1 (HIV-1) integrase protein failed to identify mutations that only blocked the integration of viral DNA and established that integrase mutations may suffer defects in virion precursor polypeptide processing, virion morphology, or viral DNA synthesis. The integrase protein is initially synthesized as a part of a gag-pol precursor polyprotein, possibly accounting for the effect of integrase mutations on the other enzymatic functions encoded by the pol gene (19, 21, 23). In an attempt to identify integrase changes that specifically prevent the integration of viral DNA, additional point mutations affecting highly conserved amino acid residues of the HIV-1 integrase protein were constructed and analyzed for their effects on viral replication in cell culture. Five HIV-1 * Corresponding author. t Permanent address: Istituto Superiore di Sanita, Rome, Italy.